Candida albicans, one of the most prevalent human pathogenic fungi, usually act as a commensal on skin and superficial mucosal, but has been found to cause candidiasis in immunosuppression patients. Previously, we have revealed the up-regulation of A-to-I RNA editing activity in response to different types of influenza virus infections. The current work is designed to study the effect of microorganism/fungi infection on the activity of A-to-I RNA editing in infected hosts.Adenosine-to-Inosine (A-to-I) RNA editing is catalyzed by the adenosine deaminase acting on RNA (ADAR) family of enzymes, which induces alterations in mRNA sequence. It has been shown that A-to-I RNA editing events are of significance in the cell’s innate immunity and cellular response to viral infections. However, whether RNA editing plays a role in cellular response to microorganism/fungi infection has not been determined. C. albicans infection. Eighty nine thousand six hundred forty eight and 60,872 A-to-I editing sites were detected in normal OKF6 and HUVEC cells, respectively. They were validated against the RNA editing databases, DARNED, RADAR, and REDIportal with 50, 80, and 80% success rates, respectively. While over 95% editing sites were detected in Alu regions, among the rest of the editing sites in non repetitive regions, the majority was located in introns and UTRs. The distributions of A-to-I editing activity and editing depth were analyzed during the course of C. albicans infection. While the normalized editing levels of common editing sites exhibited a significant increase, especially in Alu regions, no significant change in the expression of ADAR1 or ADAR2 was observed. Second, we performed further analysis on data from in vivo mouse study with C. albicans infection. One thousand one hundred thirty three and 955 A-to-I editing sites were identified in mouse tongue and kidney tissues, respectively. The number of A-to-I editing events was much smaller than in human epithelial or endothelial cells, due to the lack of Alu elements in mouse genome. Furthermore, during the course of C. albicans infection we observed stable level of A-to-I editing activity in 131 and 190 common editing sites in the mouse tongue and kidney tissues, and found no significant change in ADAR1 or ADAR2 expression .We first detected and characterized the A-to-I RNA editing events in oral epithelial cells (OKF6) and primary human umbilical vein endothelial cells (HUVEC), under normal growth condition or with C. albicans infection of human epithelial and endothelial cells led to the up-regulation of A-to-I editing activities, through a mechanism different from that of viral infections in human hosts. However, the in vivo mouse model with C. albicans infection did not show significant changes in A-to-I editing activities in tongue and kidney tissues. The different results in the mouse model were likely due to the presence of more complex in vivo environments, e.g. circulation and mixed cell types.This work represents the first comprehensive analysis of A-to-I RNA editome in human epithelial and endothelial cells. The online version of this article (10.1186/s12864-017-4374-2) contains supplementary material, which is available to authorized users. A-to-I RNA editing is a hydrolytic deamination reaction at the C6 position of adenine base occurring on double stranded RNAs, which is catalyzed by the adenosine deaminases acting on RNA (ADAR) family of enzymes , 2. InosRoles of A-to-I RNA editing were shown to be involved in innate immune response in virus-infected hosts, which displayed either antiviral or proviral activities by inducing changes that affect how viruses interact with their hosts , 14. TheCandida albicans is one of the most prevalent human pathogenic fungi. It grows as yeast or filamentous cells and can cause the infection candidiasis in humans, which was reported as the 4th leading cause [C. albicans in human hosts [C. albicans and the host immune response [C. albicans growing and coexisting with a human host without causing any symptoms of disease, is very much dependent on the immunological cross-talk between C. albicans and the human innate immune system. Alterations in the host can replace commensal factors with virulence attributes once the pathogenicity is induced. As a result, fungal cells adhering to the host epithelial cells begins invasion by host endocytosis, then active penetration and directs damage follows. Whether RNA editing plays a role in C. albicans-host immune system cross talk as a part of the innate immune response system remains unknown. In the current study, to investigate the A-to-I RNA editing events in C. albicans infections, a big-data analysis was performed using community RNA-seq data sets generated from human endothelial cells and oral epithelial cells during in vitro infection with 2 C. albicans stains, WO1 or SC5314 and from tongue and kidney tissues from mouse model of hematogenously disseminated candidiasis (HDC) [C. albicans infection was implicated with in vitro infection data.ng cause  of bloodng cause , 22. Manresponse –27. The is (HDC) . This woC. albicans infection on RNA editing of human epithelial and endothelial cells, we first analyzed the RNA-editing activities in human epithelial cells (OKF6 cell line) and endothelial cells (HUVEC cell line), which have no reported studies to date. We had 487 and 300 million paired-end RNA-seq reads (2X 100 bp) for OKF6 and HUVEC, respectively, for which the mapped reads were 409 and 268 million  and primary human umbilical vein endothelial cells (HUVEC cell line) infected with SC5314 or WO1 strains and collected at 1.5, 5, and 8 h post infection . The coneq reads X 100 bp We applied a pipeline modified from previous studies –31 to idAs shown in Fig. Comparison of the editing sites identified in two cell types Fig. , 22,741 C. albicans infection on A-to-I RNA editing in human epithelial and endothelial cells, we investigated the changes in editing activities during the course of infection. When mean editing level was used to measure the changes of editing activity  [Being the most prevalent human pathogenic fungi, is (HDC) .From our analysis, 89,648 and 60,872 A-to-I RNA editing events were identified for OKF6 and HUVEC cells, respectively. This represents the first comprehensive analysis of RNA editing editome in human epithelial and endothelial cells. The validity of these events was confirmed by cross-checking against the annotated RNA editing databases, DARNED, RADAR, and REDIportal. The significant fraction of newly identified A-to-I RNA editing events are interesting, as they represent editing sites specific to human epithelial and endothelial cells. Previously, functions of A-to-I RNA editing mediated by ADARs were discovered and documented mostly in neuronal receptors , and ionC. albicans infection, we measured their activities using the mean editing level of all events and the normalized editing levels of common editing sites across all time points. While the former displayed a slight upward trend of RNA editing activity during the course of infection, the latter showed a significant increase of RNA editing levels for sites located in the three different regions, Alu, Nonre, and Renonalu  under accession number GSE56093 and GSE67688 [C. albicans were as described [RNA-seq data for GSE67688 , 44. Celescribed , 44. Prehttps://broadinstitute.github.io/picard/). Unmapped reads and those with mapping quality score lower than 20 were removed by Samtools  [For RNA-seq data processing and mapping, our pipeline was modified from previous work –31, 45, 4 –q 20) .The protocol we used to call A-to-I RNA editing sites was derived from that described previously. In brief, the RNA-seq data mapped to reference genomes were subject to variant calling by the GATK analysis tool . The calEditing level is defined for an editing site as the number of reads with the edited base divided by the number of total reads mapped on the site. Normalized editing level is defined as the ratios of the editing levels at different time points to the maximum editing level for a specific editing site .We used CAVA  (that prLevel of expressed genes in RPKM (Reads Per Kilobase per Million mapped reads) was estimated from RNA-seq mapping results as described . BrieflyR package Venn Diagram  was usedAdditional file 1:The data sets information of collected RNA seq sample. (XLS 36 kb)Additional file 2:RNA editing sites of OKF6 and HUVEC cell lines. (XLS 29 kb)Additional file 3:Mouse organ or tissues RNA-seq mapping results. (XLS 13269 kb)Additional file 4:Cell line RNA-editing Sites overlap with database. (XLS 27 kb)Additional file 5:The number of RNA editing sites in OKF6 and HUVEC infection with different time point and strains. (XLSX 9 kb)Additional file 6:Editing level of RNA editing sites in OKF6 and HUVEC infection with different time point and strains. (XLS 28 kb)Additional file 7:Editing level of RNA editing sites locate in stable expression genes. (XLSX 9 kb)Additional file 8:Editing level of RNA editing sites locate in differentially expression genes. (XLSX 8 kb)Additional file 9:Correlation test between the genes expression of ADAR and the normalized editing level of common editing sites. (XLS 30 kb)Additional file 10:Mouse organ or tissues RNA-seq mapping results. (XLS 36 kb)Additional file 11:RNA editing sites of mouse organ or tissues. (XLS 215 kb)Additional file 12:C. albicans. (XLS 26 kb)The number of RNA editing sites with mapped bases in mouse model infection with Additional file 13:Distribution of RNA editing level of mouse organ or tissue. (XLS 27 kb)Additional file 14:Correlation test between the genes expression of Adar and the normalized editing level of common editing sites in mouse. (XLS 28 kb)


Different types of prosthesis are available for stapes replacement. Because there has been no published report on the efficacy of the titanium soft-clip vs the fluoroplastic Causse loop Teflon piston, we compared short-term hearing results of both types of prosthesis in patients who underwent stapedotomy due to otosclerosis.A total of 57 ears were included in the soft-clip group and 63 ears were included in the Teflon-piston group. Pre-operative and post-operative air conduction, bone conduction, air-bone gaps, speech discrimination score, and speech reception thresholds were analyzed.Post-operative speech reception threshold gains did not differ significantly between the two groups (P=0.919). However, better post-operative air-bone gap improvement at low frequencies was observed in the Teflon-piston group over the short-term follow-up . Similar post-operative hearing results were observed in the two groups in the short-term. The first stapes surgery was performed by Kessel in 1842 . The chaThis single-blind, randomized clinical study was carried out on 148 ears among 110 patients. All patients underwent stapedotomy between October 2013 and March 2015 at Dasthgheib Hospital, affiliated with Shiraz University of Medical Sciences. All procedures were performed by the senior author. The research protocol was approved by Shiraz University Ethics Committee (RCT code: IRCT20140121 15496N5). Inclusion criteria were patients who suffered from otosclerosis and underwent primary stapedotomy in whom we used a fluoroplastic Causse loop piston , or a titanium soft-clip . Exclusion criteria were revision stapedotomy, stapedectomy due to other causes such as congenital stapes fixation, tympanosclerotic plaque, chronic otitis media, or trauma. In addition, patients who had inadequate follow-up (less than 6 months) were excluded from our study.Patients were divided into the two groups by block randomization according to type of prosthesis. All operations were performed using a transcanal approach under local anesthesia. The footplate was drilled to a size of 0.7 or 0.6 mm by a powered microdrill for the Teflon-piston and soft-clip prostheses, respectively. The size of the Teflon-piston prosthesis was 4–6 mm in length and 0.6 mm in diameter and the soft-clip prosthesis was 4.25–4.75 mm in length and 0.5 mm in diameter. The site of the vestibulectomy was sealed with lobular fat.In order to evaluate hearing results, pure tone audiometry at frequencies of 0.25, 0.5, 1, 2, and 4 kHz was analyzed. The audiologist who reviewed the hearing results was blind to the type of prosthesis used. As we usually do not evaluate the 3-kHz frequency in our center, we used the mean of 2- and 4-kHz frequencies instead of the 3-kHz frequency. In addition, pre-operative and post-operative air conduction (AC), bone conduction (BC), air-bone gaps (ABG), speech discrimination score (SDS), and speech reception thresholds (SRT) were analyzed. A pre-operative audiogram was performed 1 week prior to the surgery, and post-operative audiograms were analyzed at least 6 months after the surgery. SPSS software version 15 was used for statistical analysis. In order to compare categorical data we used the Chi-Square test. In addition, a paired t-test or Wilcoxon test was used to compare variables within the groups and an independent sample T-test or Mann-Whitney test was used to compare between-group variables. A p-value less than 0.05 was considered significant. Initially, 148 ears which had undergone stapedotomy were evaluated. As Eleven ears in the soft-clip and four ears in the Causse loop group were excluded. The Causse loop piston prosthesis was used in 63 stapedotomies. In this group, there were 32 (50.8%) men and 31 (49.2%) women, with a mean age of 34.1±11.2 years. The soft-clip prosthesis was used in 57 cases. This group consisted of 20 (35.1%) males and 37 (64.9%) females, with a mean age of 31.2±8.4 years. The two groups were homogenous with regards to gender (P=0.08) and age (P=0.114).As We also compared mean BC gain at frequencies of 0.5–3 kHz between the two groups. Mean BC gain did not differ statistically between the groups (P=0.108) . HoweverSignificant improvement in post-operative ABG at frequencies of 0.5–3 kHz was seen in both groups (P<0.000). The improvement was greater in the Teflon-piston group in comparison with the soft-clip group (16.3 dB vs. 12.6 dB), but this difference was not statistically or clinically significant (P=0.060). ABG improvement was both clinically and statistically greater in the Teflon-piston group at frequencies of 0.25 and 0.50 kHz  .Mean post-operative ABG at 0.5–3 kHz was categorized into four groups: ≤10, 11–20, 21–30,>30 dB. As shown in Pre-operative and post-operative SDS and SRT are shown in Furthermore, the improve- ment in post-operative SDS was not statistically significant between the two groups (P=0.747).In this randomized clinical trial, we compared hearing results between the Teflon piston and the soft-clip. To the best of our knowledge, no other study has specifically compared the Teflon-piston with a titanium soft-clip. We found minimal difference between the two types of prosthesis, except for better post-operative ABG at a 0.25- and 0.50-kHz frequency in the Teflon-piston group. With respect to post-operative ABG within 10 dB, our results are within the range reported in the literature, which showed a 23–96% success rate .Both prostheses used in this study are non-crimping. Crimping is one of the most difficult stages in stapes surgery, with unpredictable results ,14,16. IEven though the soft-clip prosthesis does not need manual crimping, the narrow opening at the anterior end of its loop means that it does need to be clicked onto the long process of the incus with a gentle push. Hence, if the force applied by the surgeon is excessive, it may dislocate the incus, leading to serious consequences in terms of hearing results. In contrast, due to the nature of Teflon, its loop spontaneously returns back to its original closed shape.Thanks to modern technology, with the introduction of new biocompatible prostheses in the field of otology, otologists have the opportunity to trial various types of device; although there is conflicting evidence in the literature with regards to the superiority of one prosthesis over another. As shown in The clinical finding of this study is that there is no difference between the soft-clip and fluoroplastic Causse loop piston with regards to post-operative ABG. One of the strengths of the current study is that all patients underwent the procedure by the senior author; hence, differences in the level of expertise may be excluded as a confounding factor. The second strength of this study was the prospective and randomized methodology employed. The only major drawback of this study was its short follow-up period.We found out that both the self-crimping fluoroplastic Causse loop piston and the soft-clip, which does not need manual crimping, yielded similar post-operative hearing results in the short-term. We conclude that, if an acceptable post-operative hearing result can be achieved with the appropriate expertise, it is not necessary to pursue the latest and most expensive types of prosthesis.


Mesoporous materials enhance the solubility of PA-824; however, this is not translated into a higher antibacterial activity in TB-infected macrophages after 5 days of incubation, where similar values are obtained. The lack of improved activity may be due to insufficient release of the drug from the mesopores in the context of the cellular environment. However, these results show promising data for the use of mesoporous particles in the context of oral delivery with expected improvements in bioavailability.Tuberculosis is a major problem in public health. While new effective treatments to combat the disease are currently under development, they tend suffer from poor solubility often resulting in low and/or inconsistent oral bioavailability. Mesoporous materials are here investigated in an  According to the World Health Organization (WHO) more than 2 billion people are infected by Mycobacterium tuberculosis (MTB), and 1.3 million people died from TB in 2012 [oniazid 2 mg/kg [e [et al. , it was et al. [Although PA-824 has a high anti-tubercular activity, it suffers from poor water solubility that limits the bioavailability and limits the formulation to non-oral administrations. A traditional solution for poor solubility issues is to increase the surface area of the drug crystals by reducing their size through for instance micronization, or using solubilizers such as surfactants. However, these methods are typically not economically viable or not efficient enough in enhancing the solubility of the drug via a supersaturated state, and hence its bioavailability . Li et aet al. ,10. The et al. .et al. [M. tuberculosis-infected macrophages. Both material designs were significantly more effective than an equivalent amount of free drug in their assay. Mesoporous nanpparticles capped by ε-poly-l-lysine cationic polymer have been reported to enhance the efficacy of antimicrobial drugs [in vitro and in vivo to: load large pay loads of single or multiple active molecules [Mesoporous silica have been reported to enhance the solubility of several poorly water-soluble drugs with a variety of physico-chemical profiles ,13,14, met al. , utilizeal drugs . Mesoporal drugs . Orderedolecules ; tailor olecules ; target olecules ; increasolecules ; act as olecules ; act as olecules ; and enholecules .2/g), large pore volumes (~1 cm3/g), sharp and controllable pore size distributions, their chemically and thermally stable compositions and the low associated toxicity [et al. [There are a number of inherent physical properties of mesoporous materials which make them attractive for drug delivery applications: their high surface area  as a drr assays  on MTB-i2/g  and calculated from the decomposition temperature between 200 and 900 °C see . The mea2/g after loading. The pore volume decreases from 1.07 to 0.55 cm3/g, which indicates that more than half of the pore space is occupied by PA-824. The BET surface area of encapsulated moxifloxacin decrease from 894 to 534 m2/g, and the total pore volume change to 0.648 cm3/g from 1.07 cm3/g. A comparison of the physical adsorption properties of encapsulated and calcined materials is shown in The BET surface area of mesoporous silica particles decreases from 894 to 513 mIn vitro dissolution tests were performed in phosphate buffered saline buffer (PBS) at pH 7.4 (ca. 8 mg/L). No recrystallization occurs during the dissolution experiments.t pH 7.4 . Due to The curves of free PA-824 can be fitted for both power law and the Higuchi models  27,28,2,228,29. PA-824 against both multidrug-resistant and non-replicating MTB suggest that it may act by the inhibition of protein and lipid synthesis. The inhibition of an enzyme or deplete a cofactor that is responsible for the oxidation of hydroxymycolate to ketomycolate  may be the mechanism of PA-824 . Anotheret al. [To evaluate the intracellular efficacy of free and encapsulated drug, an assay was performed on Raw 264.7 macrophages expressing green fluorescent protein (GFP) and infected with MTB. In a previous study by Witasp et al. , AMS-6 p50  values for compounds studied are 0.40, 1.03, 0.31 and 0.88 µg/mL for PA-824, AMS-6-PA824, moxifloxacin and AMS-6-Moxi, respectively.Wells pre-plated with 0.5 mL of drug compounds, drug-loaded mesoporous silica particles or controls were dispensed with 10% heat-inactivated fetal calf serum supplement onto Raw 264.7 cells infected with M. tuberculosis (H37Rv-GFP) at a multiplicity of infection of 2:1 and dispensed into 384-well plates. After 5 days of incubation, macrophages were stained and measured for bacterial load and macrophage number . translational effect. Firstly, in the conditions of the cellular assay, the formation of protein coronas around the particles are likely to form, as has been found in a number of studies [in vivo study, where drug solubility may play a larger role [Despite a significant enhancement in the solubility of PA-824 under kinetic release studies, overall evidence of an enhanced intracellular activity is not observed for the encapsulated drugs. Several reasons could be responsible for this lack of  studies ,32, whic studies . PA-824  studies . In a ph studies . Therefoger role . As expeN-Lauroyl-L-Alanine , was first dissolved in water, followed by addition of 3-aminopropyl triethoxysilane  which was used as a co-structure directing agent and tetraethyl orthosilicate  as silica source. All chemicals were used as received. In a typical synthesis, a homogenous solution of C12Ala in distilled water is kept at 80 °C for 24 h under static conditions. The surfactant solution was stirred for 10 min before addition of APES; TEOS was added 3 min after APES. The solution was stirred for another 15 min at 80 °C in a closed bottle. The synthesis gel was subsequently stored at room temperature (RT) under stirring for 24 h. The final synthesis mixture was kept sealed at 100 °C under static conditions for 3 days. The solid product was filtered and dried at RT and under atmospheric pressure conditions. The molar composition of the reaction mixtures was C12Ala:APES:TEOS:H2O = 1:1.25:6.7:309.1. Materials were calcined at 550 °C for 6 h. The materials were stored in airtight containers after calcination.The method used for AMS-6 synthesis has been described previously . The surThermogravimetric analysis  has been using to determine the loading amount of the encapsulated samples. The scanning was performed from 20 to 900 °C at a heating rate of 20 °C/min. The plug in gas atmosphere is dry air (flow rate is 20 mL/min). The weight of the samples is varied from 5 to 10 mg. The derivative weight loss was calculated by Pyris-instrument managing software.Nitrogen adsorption-desorption isotherm  was used to investigate the porosity of calcined AMS-6 particles and the encapsulated particles. Calcined AMS-6 particles were degased at 300 °C for 6 h under nitrogen gas flow, and 8 h at 30 °C for loaded AMS-6. The surface area is calculated by the Brunauer-Emmett-Teller equation in the relative pressure range between 0.05 and 0.2 .Phosphate buffered saline (Sigma-Aldrich) used as a buffer was prepared by dissolving 1 PBS tablet in 200 mL water to give a pH of 7.4.PA-824 is a free sample from TB Alliance , and moxifloxacin is a free sample from GlaxoSmithKline . Drug compounds were loaded within the mesoporous silica particles via an evaporation technique followed by removal of the solvent using rotary evaporation. Typically, a concentrated solution of the drug was obtained in ethanol followed by direct addition of the calcined and dried mesoporous silica (200 mg) AMS-6. The slurry was stirred for 2 h, followed by solvent evaporation. Removal of the solvent was conducted at 45 °C under vacuum, before the recovered powder was dried overnight in air.in vitro release performance of drug loaded in ordered mesoporous silica was assessed in PBS buffer at pH 7.4. Five milligrams or equivalence of drug are added into 500 mL PBS buffer under a stirring rate of 100 rpm. All the release experiments showed good reproducibility. The release profile is obtained by means of UV-Vis absorbance scan .The The assay was performed as previously described . Brieflyin vitro kinetic release studies. In the present intracellular assay, mesoporous silica showed similar antibacterial activity as the free drug after 5 days of incubation in macrophage TB-infected cells. Mesoporous silica on its own did not show any antibacterial properties. The lack of improvement in antibacterial activity is likely due to insufficient release of the drug from within the pores and the lack of kinetic release results. The intracellular kinetic release of PA-824 after the particles have been engulfed by macrophages is of great interest and worth to investigate, which is also relevant for other diseases involving intracellular delivery. Further work will focus on understanding in more depth the kinetic effects associated with macrophage uptake and release through the use of fluorescent probes, as well as in the in vivo confirmation of antibacterial activity in suitable animal model and through oral administration, where mesoporous AMS-6 particles are likely to show direct benefit due to improvements in the bioavailability in comparison to the free PA-824 drug.Encapsulation of PA-824 into mesoporous particles AMS-6 results in an enhancement in the solubility and rate of PA-824


Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease. The heme peroxidase myeloperoxidase (MPO), a neutrophil-resident bactericidal enzyme, uses hydrogen peroxide to oxidize chloride and thiocyanate to hypochlorous and hypothiocyanous acids, respectively , 2. BromThe biosynthesis MPO involves a complex series of proteolytic processing and trafficking events. Initially, a single primary 80 kDa translation product is expressed in promyelocytes in human bone marrow . SubsequMPO-catalyzed reactions are thought to be involved in all stages of cardiovascular disease, from the initial development of endothelial dysfunction through to the advancement of a mature atherosclerotic plaque and finally its rupture (reviewed in ). MPO-deet al. also demonstrated the presence of a precursor form in plasma using immunoprecipitation and immunoblot analysis -1-propanesulfonate) was added to a final concentration of 1% and rotated for 2 h at 4°C. The supernatant was obtained by centrifugation at 20000 g for 20 min at 4°C.HL-60 cells were cultured and lysed as described previously with the following modifications . Cells wRabbit polyclonal anti-human MPO antibodies were produced in-house and precipitated from antiserum by adding saturated ammonium sulphate solution  at 4°C, the pellet was re-suspended in water and dialyzed into PBS. Purified human MPO was covalently coupled to NHS-Sepharose 4 Fast Flow beads following manufacturer’s instructions (0.125 mg of MPO/ml medium). Four ml of the above immunoglobulin fraction was loaded onto 2 ml of MPO-Sepharose and incubated by mixing on rotator overnight at 4°C. The medium was centrifuged at 200 g for 2 min, washed with PBS, eluted with 0.9 ml of 0.1 M glycine, pH 3.0 and the pH was neutralized with 0.1 ml 1M Tris-HCl, pH 8.0. Eluted fractions with an absorbance at 280 nm of 0.03 to 0.5 were combined and buffer-exchanged into PBS using AMICON filters with a 10 kDa molecular weight cut off . Immobilisation of the purified anti-MPO antibodies on NHS-activated Sepharose 4 Fast Flow beads (about 1 mg of anti-MPO to 2 ml of medium) was performed as above, and used for isolation of immune-reactive MPO from plasma. One ml of plasma diluted in PBS (1:2) was added to 400 μl of washed anti-MPO-Sepharose medium and incubated on a rotator overnight at 4°C. The medium was washed with PBS and MPO was eluted three times by adding 575 μl of 0.1 M glycine, pH 3.0 and neutralized with 25 μl 1 M Tris-HCl, pH 8.0. Eluates were combined and proteins were precipitated by adding 20% [v/v] trichloroacetic acid/ 20% [v/v] acetone/ 0.1% [w/v] sodium deoxycholate. Air-dried precipitates were re-suspended in reducing SDS-PAGE loading buffer , heated at 95°C for 5 min and then separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes and incubated with rabbit anti-MPO antiserum produced in-house followed by goat anti-rabbit IgG conjugated to horseradish peroxidase for visualization by the ECL detection system.SSGcAYQDVGVTcPEQDK  and MPO-specific peptide VTcPEQDKYR were detected using the MS1/MS2 transition pairs 1000.9 /1033.5 and 648.8 / 935.5, respectively and the peptide present in both proteins RNGFPVALAR using 551.8 / 272.2 (MS1 = m/z for the doubly charged precursor species and MS2 = m/z for the singly charged y ion fragment). Additionally, full CID-MS/MS spectra were acquired from m/z 200 to 2000 for the precursors (MS1). The window for precursor selection was 1 Da on either side of the m/z; the normalized collision energy was 35 and activation time 30 msec. The longer mature MPO-specific tryptic peptide, VTCPEQDKYR was chosen for SRM analysis over the shorter VTCPEQDK peptide as the latter was not detected.MPO standards or air-dried precipitates obtained after affinity-chromatography purification of plasma samples were solubilized in 100 μl of 6 M guanidine hydrochloride (GuHCl) in 25 mM bicarbonate buffer, pH 8.0 and 5 μl of 200 mM diothiothreitol was added and incubated at 57°C for 1 h. Twenty-five μl of 200 mM IAM was added and incubated in the dark for 30 min at 22°C. GuHCl was diluted to 0.6 M with 25 mM bicarbonate buffer and trypsin  was added at a ratio of approximately 1/20 (trypsin/protein) and incubated at 37°C overnight. Samples were dried and adjusted to a volume of 60 μl with water containing 0.1% formic acid. Fifty μl of sample was loaded onto an Aries Peptide XB C-18 HPLC column  for HPLC-MS analysis using a Surveyor HPLC system . An acetonitrile gradient from 98% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) was run over 25 min at a flow rate of 200 μl/min. After each gradient, solvent B was held at 100% for 5 min followed by column equilibration over 10 min with 98% solvent A. The HPLC was coupled inline to an electrospray ionization source of a Velos Pro mass spectrometer . Voltage was 4 kVolts and nitrogen gas flow was 15 AU. The temperature of the heated capillary was 275°C and vaporizer temperature was 400°C. MRM data were acquired in positive mode between 3.5 and 30 min of each chromatographic run for a pro-MPO and MPO-specific peptides as well as a peptide present in both proteins. The pro-MPO-specific peptide In some cases, immunoprecipitates were separated by 10% SDS/PAGE under non-reducing conditions as described above. After staining with Coomassie Blue, the 90 kDa band expected to contain pro-MPO was excised from the SDS/PAGE gel and subjected to in-gel digestion with trypsin at a ratio of 1/20 w/w at 37°C overnight. Tryptic fragments were eluted from the gel matrix using 80% acetonitrile, 0.1% FA, dried and taken up in 60 μl water containing 0.1% FA and analyzed by LC-MS/MS as described above.2O2/halide system, which is known to produce halohydrins [Others have shown that pro-MPO oxidizes chloride and bromide at similar rates to the mature enzyme . We wantohydrins . RecombiTo isolate MPO from human plasma, we purified MPO-specific antibodies from rabbit antiserum produced in-house and immobilized the antibodies on Sepharose. First, we tested whether anti-MPO-Sepharose could isolate pro-MPO and mature MPO from control plasma spiked with purified human mature MPO and HL-60 cell lysate containing both mature and pro-MPO  Fig 2A)Fig 2A). Next, we tested whether neutrophils release pro-MPO when added back into control plasma and stimulated with FMLP. While we observed an increase in the intensity of the mature MPO band, pro-MPO was not released into plasma by stimulated neutrophils .We then used anti-MPO-Sepharose to purify immune-reactive MPO from plasma from patients with myocardial infarction and from healthy controls . Bands cTo confirm that the 90 kDa band observed in immunoblots corresponds to pro-MPO and that it is elevated in patient samples, we developed an LC-MS/MS method for the detection of a tryptic peptide specific for pro-MPO, mature MPO as well as a peptide present in both species. The peptide amino acid sequences of the chosen peptides and their location within the MPO sequence is shown in Using the SRM-based LC-MS/MS method, we investigated the recovery of the pro-MPO-, mature MPO-specific and the mutual peptide after affinity purification and tryptic digestion of plasma spiked either 90% MPO and 10% pro-MPO or 10% MPO and 90% pro-MPO . The LC-Next, we wanted to confirm by in-gel tryptic digestion that the protein migrating at the molecular weight of pro-MPO (90 kDa) observed in immunoblots of affinity-purified plasma samples contained the pro-MPO-specific peptide. However, protein staining of the corresponding SDS/PAGE gel with coomassie did not show a 90 kDa band indicating that the amount of protein present in the gel was below the limit of detection of our mass spectrometer. Due to limited sample availability, we were unable to scale up the affinity-purification of clinical plasma samples. In contrast, control plasma samples were obtained fresh for the purpose of this study, and we were able to increase the amount of control plasma used for affinity-purification by 25-fold. We obtained a corresponding 90 kDa coomassie-stained band and in-gel tryptic digestion showed it to contain the pro-MPO specific tryptic peptide .To circumvent likely losses of protein associated with SDS/PAGE and in-gel tryptic digestion, we also performed in-solution digests of immunoprecipitates . Using tIn the present study we show by MPO affinity-purification of plasma with subsequent immunoblot and LC-MS/MS analyses that pro-MPO, an enzymatically active precursor of MPO is present in circulation. Our data further shows that circulating pro-MPO, like MPO, is elevated in patients with cardiovascular disease.Prior to this work, only one study has suggested the presence of pro-MPO in circulation when a 90 kDa band was observed in immunoblot analysis of affinity-purified plasma . TherefoHere we show that pro-MPO has bromination and chlorination activity similar to that of the mature enzyme. A kinetic study previously compared pro-MPO and mature MPO purified from human blood using multi-mixing stopped flow technique, in which the redox intermediates of these two enzymes were found to be indistinguishable . A recenThe source of circulating pro-MPO is elusive and needs further study. Our data suggests that the release of pro-MPO into plasma is independent of neutrophil activation and degranulation as activated neutrophils did not release pro-MPO into plasma. Also, immunoblots of plasma obtained from the same person on different occasions showed that the ratio of pro-MPO to mature MPO is not constant indicating alternative mechanisms for the release of these two enzymes into circulation. MPO is only actively synthesized by promyelocytes and promyelomonocytes during the differentiation process of granulocytes in the bone marrow when azurophilic granules are formed , 30. It Plasma and serum levels of mature MPO correlate well with the total number of neutrophils in the blood indicating that circulating neutrophils are the main source for mature MPO , 32. HowHere, we demonstrate that pro-MPO is elevated in patients with myocardial infarction indicating its prognostic potential that warrants further study. Pro-MPO in plasma from the cardiovascular patients might also stem from tissue macrophages, the prominent cell type in atherosclerotic lesions, where MPO has been shown to be expressed . Under nFinally, we need to acknowledge the limitations of our study. The clinical samples used for the present work were used for earlier studies and had been stored at -80°C for up to five years and freeze-thawed two to three times prior to this work. Therefore, proteins may already have undergone some degradation. Furthermore, the sensitivity of the ion trap mass analyser used for this study is low and the amount of plasma used for the affinity purification could not be increased due to limited sample availability. Collectively, this may explain the very low signal intensity when analysing immunoprecipitates by SRM-based LC-MS/MS and the fact that only a small number of y-ions were detected when full CID-MS/MS spectra were collected. Pro-MPO was detected in a greater percentage of patient samples by immunoblot analysis than by LC-MS/MS indicating the superior sensitivity of the immunochemical method. Consequently, our present data does not allow for any quantitative comparisons between control and disease groups due to the low sensitivity of the LC-MS/MS method, the small number and quality of the patient plasma samples. However, we consider the present study a proof-of concept study that warrants recruiting a greater number of samples using a more sensitive mass spectrometer like a contemporary triple-quad.Our study shows that a precursor of MPO, pro-MPO, is present in circulation and elevated in patients with cardiovascular disease. Since enzymatically active, pro-MPO can catalyse the oxidation of proteins and lipoproteins thereby fuelling the progression of an atherosclerotic lesion independent of neutrophil activation and the release of mature MPO from the azurophilic granules. Furthermore, circulating pro-MPO has the potential to be used as a marker of increased bone marrow activity. We hope that these results will stimulate further studies into the role of this enzyme plays in circulation.


However, lasso-type penalties have an obvious disadvantage, that is, the sparsity produced by them is too dispersive.Traditional drug identification methods follow the “one drug-one target” thought. But those methods ignore the natural characters of human diseases. To overcome this limitation, many identification methods of drug-pathway association pairs have been developed, such as the integrative penalized matrix decomposition (iPaD) method. The iPaD method imposes the L2,1-iPaD to identify paired drug-pathway associations. In the L2,1-iPaD model, we use the L2,1-norm penalty to replace the L1-norm penalty since the L2,1-norm penalty can produce row sparsity.Therefore, to improve the performance of the iPaD method, we propose a novel method named L2,1-iPaD method to the CCLE and NCI-60 datasets, we demonstrate that the performance of L2,1-iPaD method is superior to existing methods. And the proposed method can achieve better enrichment in terms of discovering validated drug-pathway association pairs than the iPaD method by performing permutation test. The results on the two real datasets prove that our method is effective.By applying the L Studies of the mechanism of carcinogenesis have led to the implementation that cancer is radically a disease of a variety of genetic aberrations . And at http://software.broadinstitute.org/gsea/index.jsp. Based on known gene-pathway association information, the GSEA method can forecast responsiveness of pathways. But the GSEA method does not consider the known pathway information, its identification precision is poor  and B = . We use gradient descent method ). And HΩ is an operator, and when it projects the matrix X onto the space indicated by Ω, it satisfies the following formula:Firstly, suppose that X can be expressed as follows:Hence, the optimization problem for Ω1 = 1 − Ω, which is used to indicate the missing values in the matrix Y, the problem (22) can be rewritten as:Then let The detailed proving process can be found in , 27. TheThe specific step of the optimization algorithm is summarized in Algorithm 2.λ is the only turning parameter, which is used to turn the sparsity of the drug-pathway interaction matrix B(2). The more important the drug-pathway associations is, the earlier the non-zero elements will become. Thus, we set the value of λ from producing the first non-zero elements in drug-pathway interaction matrix B(2) to 0.1. And then we use ten-fold cross-validation to obtain an appropriate λ value. Thus, we find an appropriate λ value according to the smallest residual sum of squares (RSS). The Figs. B(2) in which the values become non-zero. However, this identification method can not assess the significance of identified drug-pathway associations.In the problem (3), matrix B. The morλvalue, and calculate the P -values of every element in the drug-pathway association matrix B(2). Similar to [P-values by the following equation:B(2) in the t-th permutation. T denotes the overall number of permutations. And Therefore, we perform permutation test to assess the significance of identified drug-pathway associations after gaining an appropriate milar to , we alsoIn this section, we will show the experimental results on the real datasets, including the CCLE and NCI-60 datasets. And, in order to present the performance of our proposed method, we compare our proposed method with the iPaD algorithm.L2, 1-iPaD method, we apply this method to the CCLE data set in [L(2) is set to a zero matrix. In the iPaD method, the authors perform 2000 permutation to estimate P-values. The smaller the value of P-value is, the stronger the significance of identified drug-pathway association pairs becomes. And to be fair, we also perform 2000 permutation to assess the significance of identified drug-pathway association pairs in our method. Table L2, 1-iPaD and iPaD methods. Obviously, the L2, 1-iPaD method can mostly obtain smaller P-values than the iPaD method. In Table L2, 1-iPaD method is a sparse optimization algorithm. Thus, nonzero elements in the drug-pathway association matrix B(2) are regarded as the drug-pathway association pairs. After applying our method to the CCLE data set, we discover that the L2, 1-iPaD method can identify 368 drug-pathway pairs, whose p-values are no more than 0.05, and 66 drug-pathway association pairs among them are verified in the CancerResource. But the iPaD method can only identify 88 drug-pathway association pairs, whose p-values are no more than 0.05, and 25 pairs among them are verified in the CancerResource. And then we compute the number of identified drug-pathway association pairs that P-values are no more than 0.005. For the iPaD method, it can identify 51 drug-pathway association pairs, and 16 drug-pathway association pairs among them can be verified in the CancerResource. And our proposed method can discover 53 association pairs with 16 drug-pathway associations verified in the CancerResource database. Tables L2, 1-iPaD and iPaD methods. Note that in Table In this subsection, in order to assess the performance of a set in . CCLE daa set in . In thisL2, 1-iPaD method to the lung cancer data set, which is extracted from the CCLE data set. And we also apply the iPaD method to analyze lung cancer data set. Table We note that combining different cancer types of data can increase the number of samples and can be better to identify common signals from different cancer. But, this operation may weaken the knowledge, which is specific to certain cancer types. Thus, in this paper, we apply the http://discover.nci.nih.gov/cellminer. This data set contains 57 cell lines from eight different cancer types and 1863 genes covering 58 KEGG pathways and 101 drugs. The drug sensitivity is measured by GI50 values, which is the minimum concentration of the drug needed to inhibit the growth of 50% [GI50values can manifest the drug-sensitive response, the higher GI50 values can manifest the drug-resistant response [λ value. And then we also perform 2000 permutations to obtain the P-values, which can estimate the significance of the drug-pathway associations. Table P-values on NCI-60 data set for the L2, 1-iPaD and iPaD methods. After applying our proposed method to the NCI-60 data set, we discover that the L2, 1-iPaD method can find 562 association pairs, with the P-values no more than 0.05, and 163 pairs among them are verified in the CancerResource. However, the iPaD method can only discover 247 drug-pathway association pairs with the P-values no more than 0.05, and among those drug-pathway associations only 74 drug-pathway pairs are verified in the CancerResource. And then we calculate the number of identified drug-pathway association pairs that P-values are no more than 0.005. The iPaD method can find 72 association pairs with 26 drug-pathway association pairs among them validated in the CancerResource database. But our proposed method can identify 89 drug-pathway association pairs with 33 association pairs among them validated in the CancerResource. Tables L2, 1-iPaD and iPaD methods. From Tables We also apply our method on the NCI-60 data set, which has been used in , 13. Theh of 50% . As a coL2, 1-iPaD” to discover novel drug-pathway associations. In the optimization model, the objective function has only one turning parameter λ. Thus, our proposed method is nearly turning-free. To find the best performance of our method, we apply ten-fold cross-validation to discover an appropriate λ value. And to estimate the significance of the identified drug-pathway association pairs, we perform permutation test to calculate the P-values. For the purpose of assessing the performance of the L2, 1-iPaD method, we apply this method in the CCLE and NCI-60 datasets. The experimental results in the CCLE and NCI-60 datasets demonstrate that our proposed method can discover more drug-pathway association pairs than the iPaD method. And the L2, 1-iPaD method can identify more validated associations.Drug-pathway association identification is an important issue in pharmacology. In this paper, we develop an effective algorithm named “With the development of genomics and pharmacology, dealing with transcription and drug sensitivity data has become feasible. Our proposed method has tremendously improved the performance of the original algorithm. In the future, we are ready to propose more efficient and robust algorithms to handle the high-throughput drug related data. And the rapid growth of the high-throughput gene expression and drug related data is calling for more effective algorithms to solve the computational problems.


Giant cell tumor of bone (GCT), which frequently occurs in the patients’ spine, is relatively prevalent in Chinese population. A group of GCT invades into vessels and appears to be circulating tumor cells (CTCs) responsible for the distal metastasis of the primary tumor. So far the cell surface markers of GCT have not been determined. In the current study, we aimed to identify a novel CTC marker with higher specificity in GCT. TRAIL-R1+ cells were purified from GCT cell lines. The TRAIL-R1+ cells were compared with total GCT cells for tumor sphere formation, chemo-resistance, tumor formation in nude mice, and frequency of developing distal metastases. We found that TRAIL-R1+ GCT cells appeared to be highly enriched for CTCs in GCT. Compared to total GCT cells, TRAIL-R1+ GCT cells generated significantly more tumor spheres in culture, were higher chemo-resistant, and had a higher frequency of being detected in the circulation after subcutaneous transplantation as well as development of distal metastases. Thus, we conclude that TRAIL-R1+ may be a novel CTC marker in GCT. Selective elimination of TRAIL-R1+ GCT cells may improve the current GCT therapy. Giant cell tumors of bone (GCT) are rare bone tumors worldwide, but relatively prevalent in Chinese population . Most GCChemotherapy is an important method to assist radiotherapy for treating GCT . DenosumDuring pathological progression of GCT, a group of GCT cells may invade into vessels and become circulating tumor cells (CTCs) that had dissembled from the primary tumor and subsequently invaded into the blood circulation . The mosActivation of the TNF-related apoptosis-inducing ligand (TRAIL) pathway regulates cancerous cell apoptosis in human. TRAIL-R1 is a receptor for apoptosis ligand TRAIL. Very recently, TRAIL-R1 has been reported to play a role in the carcinogenesis –15. HoweTwo human GCT cell lines Hs737.T and Hs127.T was used in the current study. Both lines were generated from giant cell sarcoma from the bone. A lentivirus carrying both luciferase and GFP reporter under the control of a CAG promoter was used to transduce Hs737.T and Hs127.T cells, for tracing tumor formation in living mice and for isolating tumor cells from mice Figure . After tTRAIL-R1 was used to separate GCT cell population into 2 fractions, TRAIL-R1+ and TRAIL-R1- cells, by flow cytometry Figure . We checin vitro.The TRAIL-R1- and TRAIL-R1+ cells from both lines were then subjected to tumor sphere formation assay. We found that compared to TRAIL-R1- cells, TRAIL-R1+ cells generated significantly more tumor spheres in both lines, shown by representative images Figure , and by Same number of TRAIL-R1- and TRAIL-R1+ cells was subcutaneously transplanted into Nude mice, and the tumor formation was monitored after 8 weeks. We found that compared to TRAIL-R1- cells, TRAIL-R1+ cells generated significantly larger tumor shown by quantification Figure  and by tFinally, 30 tumor cells were isolated from the primary tumor developed from either TRAIL-R1- and TRAIL-R1+ cells, and were transplanted back to new nude mice. The formation of the tumor was first verified by bioluminescence and then confirmed by histology of the dissected out at 6 weeks. The newly formed tumors were then dissected out and used for isolation of 30 tumor cells for the next round of transplantation. Three rounds of transplantation were performed. We found that TRAIL-R1+ cells had significantly higher rate of tumor formation after serial adoptive transplantations, compared to TRAIL-R1- cells, based on bioluminescence examination Figure . MoreoveIn the current study, we analyzed TRAIL-R1 as a novel CTC marker for GCT. The ignition of the study was encouraged by a recent study in neural science, which shows that TRAIL receptors may play a role in the radio-resistance of human neural stem cells and neuroblastoma cells . We haveWe chose two human GCT lines in this study, since both represent giant cell tumor of the bone, but appear to be different in malignancy. Hs737.T was from a 10-year-old female, while Hs127.T was from a 64-year-old male. Hs737.T grew faster and appeared to be more aggressive than Hs127.T in culture. Thus, analysis on both lines increased the reliability of the study and the results may be more applicable to GCT.in vitro and in vivo, appeared to be more resistant to denosumab, were more frequently detected in the circulation, and generated tumor more frequently after serial adoptive transplantation. These are gold standards for determining CTC with tumor forming potentials. Indeed, here we used a set of experiments to characterize that TRAIL-R1+ GCT cells may be a fraction of circulating tumor stem-like cells (CTSC), which have properties of tumor formation, high invasiveness and chemo-resistance [We purified TRAIL-R1+ GCT cells, and compared to TRAIL-R1- GCT cells. We found that these TRAIL-R1+ cells showed higher tumor formation potential sistance . IdentifDenosumab is a FDA-approved human monoclonal antibody with high affinity and specificity to RANKL, the receptor activator of nuclear factor kappaB ligand, which is the principal mediator of osteoclastic bone resorption , 6. The Although here we provide direct data to support the usefulness of TRAIL-R1 as a surface marker for enriching CTSC in GCT, further evidence from clinical studies is required to confirm the findings. Nevertheless, here our study demonstrates that TRAIL-R1+ may be a novel CTC marker in GCT. Selective elimination of TRAIL-R1+ GCT cells may improve the current GCT therapy.All the experimental methods have been approved by the research committee at Union Hospital, Tongji Medical College. All animal experiments were approved by the Institutional Animal Care and Use Committee at Union Hospital, Tongji Medical College. All the experiments have been carried out in accordance with the guidelines from the research committee at Union Hospital, Tongji Medical College. The methods regarding animals were performed under the approval of institutional Ethics Committee.2 Denosumab  was given to cultured cells at the concentration of 10 μmol/l. The cells were analyzed 24 hours after treatment.Two human GCT cell lines Hs737.T and Hs127.T were both purchased from ATCC . These cells were cultured in Dulbecco's Modified Eagle's Medium suppled with 10% Fetal Bovine Serum . The cells were incubated in a 37°C incubator with 5% COin vivo visualization of the implanted tumor cells and for detection of the CTCs by flow cytometry. The pcDNA3.1-CAG-GFP plasmid and a pcDNA3.1-CAG-luciferase plasmid were applied in backbones . The GFP coding sequence was digested with Xhol and BamHI and subcloned with a 2A into a pcDNA3.1-CAG-luciferase, and pCAG-luciferase-2A-GFP was obtained. For constructing lentiviral particles, HEK293T cells were seeded in 100 mm dish at 50,000 cells/cm2 and co-transfected with 10 μg of pCAG-luciferase-2A-GFP and 5 μg each of packaging plasmids  with Lipofectamine-2000 (Invitrogen). The supernatant was removed 48 hours after transfection and filtered through the 0.45 μm syringe filter. The virus in supernatant was isolated and titered. For cell transduction in vitro, GCT cells were seeded in 100 mm plates at 15,000 cells/cm2 one day prior to lentiviral infection. The lentiviral particles were added along with 10 μg/ml polybrene (Sigma-Aldrich) to the cell culture at a multiplicity of infection (MOI) of 100 for 48 hours. Then the cells were rinsed twice with complete media and purified for transduced cells (efficiency of 26.7% for Hs737.T and of 9.1% for Hs127.T cells) based on GFP with flow cytometry. The transduced cells were observed in vivo with luciferase, and determined with GFP.The GCT cells transduced with lentivirus carrying Luciferase (LUC) and green fluorescent protein (GFP) reporters for Ten week-old male NOD/SCID mice  were used for subcutaneous transplantation of tumor cells and serial adoptive transfer. The bioluminescence was monitored 4 weeks after transplantation. For transplantation of cancerous cells into NOD/SCID mice, 200 cells were implanted and the tumor formation was monitored after 8 weeks by bioluminescence. For serial adoptive transplantation of cancer cells, 30 cancer cells were isolated from implanted tumor and re-transplanted back into the mice. The tumor formation was examined after 6 weeks by bioluminescence. Three rounds of serial adoptive transfer were performed.Formation of tumor was monitored by luciferin assay, based on luciferase activity of tumor cells. All the mice were anesthetized with 3% isoflurane and then luciferin (Sigma-Aldrich) of 150 mg/kg body weight were injected intraperitoneally. After 10 minutes, the bioluminescence of mices were observed and imaged with IVIS imaging system , with an acquisition time of 60-second and binning of 10. The images were processed and analyzed with the software of Living Imaging system.4 cells/plate. Then the formation of tumor sphere was observed and recorded.After cancer cells were obtained, all the cells were dispersed to single cells with enzymatic digestion. Afterward, single cancer cells were re-suspended in tumor sphere media (TSM). The TSM was a serum-free DMEM, with human recombinant Epidermal growth factor (20 ng/ml), bFGF (20 ng/ml), leukemia inhibitory factor (10 ng/ml) and N-acetylcysteine (60 μg/ml). The cells were then smeared in 60 mm petri dish at a density of 2 × 104/ml and seeded in a 96-well microplate. After 24 h, cells were treated with resveratrol. Then the CCK-8 reagents were added and incubated. The absorbance of wells in microplate was read at 450 nm with microplate reader. The absorbance value was positively correlated to cellular viability. The cell viability was calculated as: the percentage of absorbance value in detected well with reference to control well (control group without treatment was 100%).The cell viability was determined with CCK-8 detection kit (Sigma-Aldrich). First, cells were prepared with a density of 5 × 10p < 0.05 was considered as significant after Bonferroni correction.The statistical analysis was performed with the GraphPad Prism 6 . Comparison of group differences was carried out with a one-way analysis of variance (ANOVA) test and then Turkey multiple comparison post-hoc analysis. All values represent the mean ± standard deviation (SD). A value of


Although Perceived Stress Scale PSS,  has beenThe purpose of this study was to examine the measurement invariance of PSS between athletes and non-athletes, and examine construct validity and reliability in the sports contexts.Mage = 19.88 yrs, SD = 1.35) and examined discriminant validity and convergent validity of PSS. Study 3 sampled 37 student-athletes to assess test-retest reliability of PSS.Study 1 sampled 359 college student-athletes  and 242 non-athletes  and examined factorial structure, measurement invariance and internal consistency. Study 2 sampled 196 student-athletes .Results found that 2-factor PSS-10 fitted the model the best and had appropriate reliability. Also, there was a measurement invariance between athletes and non-athletes; and PSS positively correlated with athletic burnout and life stress but negatively correlated with coping efficacy provided evidence of discriminant validity and convergent validity. Further, the test-retest reliability for PSS subscales was significant (It is suggested that 2-factor PSS-10 can be a useful tool in assessing perceived stress either in sports or non-sports settings. We suggest future study may use 2-factor PSS-10 in examining the effects of stress on the athletic injury, burnout, and psychiatry disorders. Since the development of the Perceived Stress Scale PSS,  it has bBuilt on Mage = 42.8 ± 17.2 years) and examined its factorial structure, criterion validity, and internal consistency of the PSS. Results found 10-item, by deleting item 4, 5, 12, and 13, 2-factor PSS-10 can be a better measuring tool of perceived stress because they found the revised version of PSS-10 accounted 48.9% of the variance, and had better reliabilities (Cronbach’s α = .84 ∼.86), and correlated with anxiety, depression and life events which indicated good construct validity.Given that the applicability of PSS in assessing perceived stress in many domains, the number of the item, the factorial structure, and the reliability of PSS have been intensively examined by many researchers. For example, although the initial version of PSS  has beenα = .81) and perceived coping (Cronbach’s α = .72) with 11 items. Similar findings also found in a Mexico sample where Followed Cohen and colleagues Based on above literature, we understand that earlier version of one-factor 14-PSS  was desiIn sports, stress is an important issue that has been received much of attention; specifically in the studies of athletic burnout and athletic injury. For example, Many researchers borrow either stress-burnout or stress-injury concepts to examine the role of perceived stress on athletes’ burnout and injury. Researchers use different measures to do their studies. For example, To assess global perceived stress in sports, some researchers used PSS in examining its’ relationship with sports injury and burnout. For example, in examining whether stress and affect as the mediator of hope-burnout relationship, Gustafsson and colleagues In terms of stress- athletic injury relationship, Galambos and colleagues As previously mentioned, although sports researchers used either PSS-10 or PSS-14 in examining their relationships with athletic burnout or injury, the psychometric properties of PSS have never been examined in sports. Thus, there are several questions remained. First, whether two-factor or unidimensional PSS will be suitable for sports? Second, if we compare athletes and non-athletes will measurement characteristics remains the same? Third, what is the reliability and validity of PSS in sports? Therefore, the existing knowledge gap of the application of PSS in sports is that we don’t know which type of PSS is suitable in terms of dimensionality, construct validity, reliability, and group comparisons between athletes and non-athletes. To fill the gap, there are three purposes in this study. First, we intended to examine the factorial structure of the PSS-10 and PSS-14 and internal consistency. Second, we intended to examine the measurement invariance of PSS between athletes and non-athletes. Third, we attempted to examine the construct validity of PSS and test-retest reliability of PSS.The purpose of study1 was threefold: (a) to compare the factorial structure of the PSS-10 and PSS-14; (b) to examine internal consistency of PSS-10 and PSS-14; (c) to examine measurement invariance of PSS between athletes and non-athletes.We recruited 359 college student-athletes  with mean age 20.08 (SD = + 1.51) from two sport-colleges and three universities in Taiwan. At the time of data collection, athletic participants were all in their regular training seasons and had been participating in a variety of individual and team sports, such as gymnastics, track and field, golf, weightlifting, basketball, volleyball, soccer, Tae-kwon-do, badminton and baseball for 8.93 years (SD = 3.14) of training and competition experiences.Mage = 20.10 yrs, SD = + 1.55) participated in our study. We collected their data during their classes. They are all regular students studied at different academic departments such as accounting, political science, biology, mathematics...etc. They participated in our study voluntarily without any conditional requirements from the classes.For non-athlete participants we recruited 242 , the first author contacted the coach/teacher of a target team and class and asked permission to use his/her team/class as participants. Once the coach/teacher agreed to use his/her team/class as participants, we visited target team/class one hour before they finished the regular training/class. Before administering the questionnaire package, the second author explained the general purpose of the study, the method to complete questionnaires and rights of being a participant. To prevent social desirability effects, we informed participants that this is a study to explore college students’ life experiences, and there were no right or wrong answers. Additionally, we asked them to answer the questions as truthfully as possible, and all responses would be confidential. After the briefing, participants who interested in this study then signed a consent form and completed the demographic questionnaire and 14-item PSS. It took about 15 min to complete the questionnaires.The demographic questionnaire was designed to gather information about participants’ age, gender, types of sports, and years of athletic experiences.The PSS is a self-report measure designed to assess one’s perception about the degree of a given situation in daily life is considered stressful . The PSSα coefficient was used as an index.All primary statistical testing was conducted in AMOS version 22. Models of the PSS-14 and the PSS-10  were first estimated separately for the 1-factor and 2-factor as For testing measurement invariance, we adopted earlier suggestion  by follo 1.Configural invariance  also re. This st 2.Metric invariance  also re. This st 3.Factor variance/covariance invariance . This step constrained the like factor variances across the groups . If fact 4.Error variance invariance. This step constrained error variances across the groups. If the same level of measurement error is present for each item between groups. This step tested whether the error of stress and counter-stress all items were related equivalently across athletes and non-athletes.χ2 difference tests are typically used to compare nested models. However, the χ2 difference test may also be influenced by sample size  was also conducted  and .71 (counter stress).The purpose of study 2 was to examine the construct validity of 2- factor PSS-10, which is the convergent and discriminant validity, via correlational analyses surrounding the relationships among PSS-stress, PSS counter-stress, college student-athletes’ life stress, coping self-efficacy and burnout experiences.Mage = 19.88 yrs, SD = 1.35). The recruiting procedure was similar to study 1.A new sample of the targeted population was recruited. Valid data of 196 student-athletes from ten different universities were collected  to 6 . In the present study, the result of CFA confirmed that the factorial structure was suitable for the data. The Cronbach’s α for the three subscales ranged from .63 to .86 and the reliability for all items was .90. To further identify convergent validity it is expected that the burnout scale should be positively correlated with the PSS stress because athletes’ stress has been identified as a leading factor of athlete burnout (ABQ  is a sel burnout . The PSSNever) to 6 (Always). Cronbach’s α of these factors ranged from .69 to .87 and the reliability for all items was .92 in this study, indicating that the result was reliable. Given that CSALSS represents an individual’s life stress, the PSS counter-stress factor was expected to have negative with CSALSS, while PSS stress was expected to have a positive relation with CSALSS.The 24-item CSALSS  was usedα for the three subscales ranged from .70 to .78 and the reliability for all items was .86. It is expected that CSE will be negatively correlated with the PSS stress but positively correlated with PSS counter-stress.CSE  is a selχ2/DF ratio; (2) the root mean square error of approximation (RMSEA); (3) the standardized root mean square residual (SRMR); (4) the Goodness of Fit Index (GFI); (5) the Comparative Fit Index (CFI); and (6) the Non-Normed Fit Index (NNFI). The recommendations for good fit are the following: χ2/DF ratios between one and three, values for RMSEA values less than 0.08 along with SRMR values less than 0.05, and GFI/CFI/NNFI values greater than 0.90   the han 0.90 . To examCR > .7)  and averCR > .7)  were calχ2/DF = 1.607, CFI = .960, NNFI = .903, GFI = .955). The factor loadings for the 10 items range from .33 to .87 . The data was collected in 8–9 days, and with the general procedures in the previous section. However, because two participants were unable to complete all items, their data were dropped from the study.Another new sample was recruited from Judo and Boxing , and counter stress was  which indicated that two subscales significantly reliable over measuring time.Results indicated that the Pearson coefficients for perceived stress (In line with past research examining the validity of PSS the purpose of this study was to examine psychometric properties of PSS in sports settings. Specifically, this study attempted to examine factorial structure, measurement invariance, reliability, and construct validity of PSS. By three studies we found 2-factor PSS-10 has a better fit of the model. Also, we found 2-factor PSS-10 had appropriate internal consistency, testretest reliability and measurement invariance across athletes and non-athletes. Further, we found 2-factor PSS-10 positively correlated with athletes’ life stress and burnout but negatively correlated with coping self-efficacy which indicated appropriate construct validity.Therefore, the psychometric properties of PSS gain solid supports in the sports contexts. The results of factorial structure suggest that 2-factor PSS-10 has better measuring quality than 2-factor PSS-14, or 1-factor PSS-10/PSS-14. The 2-factor PSS-10 reduces four items allows researchers collect data in a short period of time . AlthougIn terms of measurement invariance, we found configuration invariance, metric invariance, and variance/covariance invariance are all equivalent except error variance invariance. Therefore, it means that the same level of measurement error for each item between athletes and non-athletes is not the same. However, A significant feature of this study is the sample recruited from Taiwanese student-athletes. Although PSS has been validated in different culture such as Spanish , SwedishFurther, as previously mentioned that most research in sports either using As to the application of 2-factor PSS-10 in other domains, we suggest it can be a useful tool in assessing perceived stress in an intervention study. For example, if researchers want to know whether a stress management program reducing participants’ perceived stress e.g., . In suchThere are several limitations that need to be addressed. First, our sample is all recruited from Division I college student-athletes whether our results could be generalized to other athletes, such as professional athletes or junior athletes, need to be further examined. Additionally, the data was collected from Taiwanese student-athletes; hence, the results may not be generalizable to different cultures. We recommend researchers adopt similar approaches to test measurement invariance in different cultures and populations.To acknowledge that PSS is a widely used measure in assessing stress, we have conducted three studies to examine the factor structure, measurement invariance between athletes and non-athletes, internal reliability, test-retest reliability and construct validity in the sports contexts. Results indicated that 2-factor PSS-10 can be an ideal measure for the research in sports. We suggest future research may use 2-factor PSS-10 in conducting various stress research.10.7717/peerj.2790/supp-1Data S1Click here for additional data file.10.7717/peerj.2790/supp-2Data S2Click here for additional data file.10.7717/peerj.2790/supp-3Data S3We collected athletes’ demographics, perceived stress, coping stress, burnout, and life stress. Results showed perceived stress and counter perceived stress related with other latent variables provides concurrent and discriminant validity as Click here for additional data file.10.7717/peerj.2790/supp-4Data S4Click here for additional data file.


To provide an up-to-date assessment of the effectiveness of the Mental Health First Aid (MHFA) training program on improving mental health knowledge, stigma and helping behaviour.Systematic review and meta-analysis.A systematic search of electronic databases was conducted in October 2017 to identify randomised controlled trials or controlled trials of the MHFA program. Eligible trials were in adults, used any comparison condition, and assessed one or more of the following outcomes: mental health first aid knowledge; recognition of mental disorders; treatment knowledge; stigma and social distance; confidence in or intentions to provide mental health first aid; provision of mental health first aid; mental health of trainees or recipients of mental health first aid. Risk of bias was assessed and effect sizes (Cohen’s d) were pooled using a random effects model. Separate meta-analyses examined effects at post-training, up to 6 months post-training, and greater than 6 months post-training.A total of 18 trials (5936 participants) were included. Overall, effects were generally small-to-moderate post-training and up to 6 months later, with effects up to 12-months later unclear. MHFA training led to improved mental health first aid knowledge (ds 0.31–0.72), recognition of mental disorders (ds 0.22–0.52) and beliefs about effective treatments (ds 0.19–0.45). There were also small reductions in stigma (ds 0.08–0.14). Improvements were also observed in confidence in helping a person with a mental health problem (ds 0.21–0.58) and intentions to provide first aid (ds 0.26–0.75). There were small improvements in the amount of help provided to a person with a mental health problem at follow-up (d = 0.23) but changes in the quality of behaviours offered were unclear.This review supports the effectiveness of MHFA training in improving mental health literacy and appropriate support for those with mental health problems up to 6 months after training.CRD42017060596)PROSPERO ( Up to 1 in 5 adults will develop a mental health problem in any year. DevelopFrom its beginnings in Australia, the MHFA program has spread worldwide with more than 22 countries adopting the program, and over 2 million people attending a course . The StaA meta-analysis of 15 studies evaluating MHFA found that the program improves mental health knowledge, reduces stigmatising attitudes, and increases helping behaviours . HoweverThis review was conducted in accordance with the PRISMA recommendations for systematic reviews and the Studies were eligible if they were randomised controlled trials (including cluster-randomised), quasi-randomised controlled trials, or controlled trials, in adults aged 18+. We excluded uncontrolled trials given their greater risk of yielding a biased estimate of effect . We inclth April 2017. This systematic search was also re-run on 12th Oct 2017, to detect any additional studies published since the initial search. We searched PubMed, PsycINFO (OVID interface), EMBASE (OVID interface), and the Cochrane Central Register of Controlled Trials (Wiley interface) for eligible studies. Specific search strategies were developed for each database, using a combination of both key words and MeSH/Map terms to include the following: ‘controlled trial’, ‘mental health’, ‘mental disorder’, ‘first aid’, ‘mental health training’, ‘MHFA’, ‘mental health first aid’, limiting to studies involving humans  and by examining the list of MHFA course evaluations provided on the MHFA International website . The reference lists of included studies and relevant reviews identified through the search were also checked. One author (AR) screened titles and abstracts for potential inclusion and two authors  independently screened the full-text of retrieved articles, with discrepancies resolved by consensus or arbitration from a third author (NJR). The flow of studies is presented in A systematic search of the literature was conducted for studies published between the year of MHFA inception (2000) and 5A data extraction template was developed and piloted. Study characteristics and outcome data from each study were extracted independently by two authors, with disagreements resolved by discussion. We attempted to contact authors to resolve any uncertainties or to obtain missing information, and received information from 10 studies –22. InfoFour sets of primary outcomes were evaluated: (1) knowledge, (2) stigmatising attitudes, (3) confidence and intentions in providing MHFA, (4) provision of mental health first aid. These outcomes are not measured as part of MHFA training but have been evaluated in research trials as they measure the main constructs of the training.It is not a good idea to ask someone if they are feeling suicidal in case you put the idea in their head and It is best not to try to reason with a person having delusions). These questions have face validity but no other psychometric properties have been reported. Recognition of mental health problems was based on accuracy of identification of mental health problems described in a short vignette, most commonly depression or schizophrenia. These vignettes were written to satisfy diagnostic criteria and correct recognition has been validated by an Australian national sample of clinical psychologists, psychiatrists, and general practitioners (GPs) [Three kinds of knowledge outcomes were extracted, where available. Mental health first aid knowledge, when measured objectively, typically by true or false questions based on course content (e.g. rs (GPs) . Beliefsrs (GPs) , 25. ForStigmatising attitudes towards people with mental health problems were primarily assessed with Social Distance Scales based on Link et al and PersConfidence and intentions to provide MHFA were extracted as proxy measures of behaviour change. Perceived confidence was usually assessed with a single question in relation to helping a person with a mental health problem. This has been shown to be predictive of high quality support provided up to six months later . IntentiThis was assessed in two ways in included studies: the amount of help or mental health first aid provided to someone with a mental health problem, and the quality of first aid provided. Studies measured amount of help either on frequency scales (e.g. “never” to “many times”) or whether help had been provided at all (yes/no). Quality was assessed by summing the number of helpful actions participants reported taking to support a person with a mental health problem, which were consistent with MHFA training.Secondary outcomes collected were:Mental health of MHFA trainees, assessed using a validated measure of depression, anxiety, or psychological distress/internalising symptoms.recipients of mental health first aid (via MHFA trainees), assessed using a validated measure of depression, anxiety, or psychological distress/internalising symptoms.Mental health of Where outcomes were reported as both continuous (e.g. means and standard deviations) and dichotomous (e.g. percentage above a cut-off), we extracted the continuous data. Where studies reported both endpoint data and change over time data (e.g. group by time interactions) we extracted the change over time data due to the potential for baseline imbalances. Where intentions, confidence or behavioural outcomes towards multiple people were reported (e.g. students AND colleagues), we extracted data for the person who was the clearly intended recipient of MHFA (i.e. school students for Youth MHFA participants).Risk of bias was assessed using the revised Cochrane Risk of Bias tool for randomised trials (RoB 2.0) . This had of 0.2 = small, 0.5 = medium, and 0.8 = large [Outcomes were analysed with Comprehensive Meta-Analysis (CMA) V2 software using a random-effects model and reported as standardized mean differences. Separate meta-analyses were conducted to examine effects at 3 different pre-specified time-points . Multiple validated outcomes of the same construct  within a study were combined into one effect size using CMA, unless these data were already pooled and reported by trial authors. Where studies reported an effect size for more than one independent subgroup in their sample (e.g. depression vignette versus schizophrenia vignette), we included each subgroup as a separate ‘study’. When interpreting mean effect sizes, we followed Cohen’s guidelines whereby  = large .To explore the robustness of the results we performed several pre-specified sensitivity analyses. We tested the impact on pooled estimates of removing studies conducted by the developers of MHFA, studies judged to have high risk of bias, and outlier studies (studies whose 95% CI did not overlap with the 95% CI of the pooled effect size).2 statistic, which expresses the amount of heterogeneity in effect sizes in percentages. A percentage of 25% indicates low heterogeneity, 50% moderate and 75% high heterogeneity [2 was calculated using the ‘heterogi’ module in STATA. Where I2 was not zero and the confidence interval included high levels of heterogeneity, further subgroup and meta-regression analyses were conducted to explore possible causes of heterogeneity. Subgroup analyses used a mixed-effects model (a random-effects model within subgroups and a fixed-effect model across subgroups) and explored whether mean effect sizes differed for face-to-face versus online delivery, standard adult course versus youth MHFA, and type of comparison condition. When there were at least 3 studies per analysis, meta-regression explored the effect of the percentage of female participants and program length.Statistical heterogeneity for each pooled outcome was examined with the Iogeneity . The 95%Small study effects (e.g. publication bias) were assessed when there were 10 or more studies in the meta-analysis by visually examining the funnel plot, supplemented by Egger’s test of funnel plot asymmetry , 40. DuvSearches generated 611 records of which 34 were assessed for full-text eligibility see . SixteenThere were 4 cluster-RCTs, 10 RCTs, and 4 controlled trials. Studies used a variety of comparison conditions, with 6 using a waitlist condition, 6 using another health or mental health education program, and 6 using a no intervention design. Outcomes were assessed post-intervention in 8 studies, up to 6 months follow-up in 13 studies (14 comparisons), and only 2 studies examined effects at more than 6 months post-intervention (up to 1 year follow-up). Three studies were unpublished or ongoing , 20, 21.Most studies evaluated the adult course or a variant (k = 15) rather than youth MHFA (k = 3) and delivered the course in a face-to-face format (k = 15) rather than online or CD-ROM (k = 3). The program was evaluated in several settings, including workplaces (k = 7), with university or heath care students (k = 5), members of the public (k = 3), teachers (k = 1), parents (k = 1), and the military (k = 1). Study participants tended to be female . The majority of studies were conducted in Australia (k = 8), with 5 conducted in North America, 4 in Europe/UK, and 1 in Hong Kong. Most studies did not involve the founders of MHFA in their evaluation (k = 12).Risk of bias was rated per outcome according to Cochrane guidelines and is sd = 0.22), with low-to-moderate heterogeneity, increasing to moderate improvements by 6-month follow-up (d = 0.52) with low heterogeneity. Effect sizes were similar with the removal of 3 studies involving the developers of MHFA at post-intervention, d = 0.39  and 4 studies at follow-up, d = 0.43 . There was no evidence of publication bias in the 11 studies at follow-up < = 6 months .As shown in d = 0.45) and at up to 6-month follow-up (d = 0.19) with moderate-to-high heterogeneity in effect sizes. Effects were robust in sensitivity analyses exploring researcher allegiance post-intervention, d = 0.91  and follow-up, d = 0.17 . Removal of one outlier study[d = 0.16 . There was no evidence of publication bias in the 11 studies at follow-up < = 6 months .Beliefs about effective treatments for mental health problems significantly improved at post-intervention (ier study at follod = 0.72), which was smaller at 6-month follow-up (d = 0.54) and 12 month follow-up (d = 0.31), with no heterogeneity. Removing studies involving the developers of MHFA increased the effect size at post-intervention to d = 0.91  and d = 0.53  at 6-month follow-up. Mean effect sizes were the same when removing studies at high risk of bias, d = 0.72  at post-intervention and d = 0.54  at follow-up less than 6 months.There was a moderate-to-large significant improvement in MHFA knowledge at post-intervention (d = 0.14) and up to 6-month follow-up (d = 0.14), reducing to very small non-significant effects at 12-month follow-up (d = 0.08). Heterogeneity was low at post-intervention and moderate at 6-month follow-up. Effects were weaker when removing studies involving the founders of MHFA at post-intervention, d = 0.09  and 6-month follow-up, d = 0.11 , but confidence interval limits overlapped. Similarly, removing one outlier study[d = 0.09 . Visual inspection of the funnel plot suggested some asymmetry in effects at 6-month follow-up . The Duval & Tweedie trim-and-fill approach suggested that four studies were potentially missing and, if imputed, the mean effect size would drop to d = 0.08 and would no longer be significant .There were small significant effects on stigmatizing attitudes at post-intervention  for social distance (k = 4) and d = 0.12  for personal stigma (k = 8). Similarly, at up to 6 months follow-up, effect sizes tended to be larger for social distance, d = 0.23  than for personal stigma, d = 0.06 .Supplementary analyses were conducted to explore whether effects varied depending on the type of stigma, as most studies included measures of both social distance and personal stigma. The mean effect size at post-intervention was d = 0.58) and follow-up by 6 months (d = 0.46), with high levels of heterogeneity. Effects beyond 6 months were small in size (d = 0.21). There were two outlier studies at 6-month follow-up [d = 0.43 . Results were robust to researcher allegiance, with very similar effects at post  and < = 6-month follow-up . However, inspection of the funnel plot for the 12 studies at 6-month follow-up suggested evidence of asymmetry . The Duval & Tweedie trim-and-fill approach suggested that four studies were potentially missing and, if imputed, the mean effect size would drop to d = 0.32 .There were moderate significant improvements in confidence in helping someone with a mental health problem at post-intervention  and by 6-month follow-up (d = 0.55 with high heterogeneity), with smaller effects observed at longer follow-up (d = 0.26 with moderate heterogeneity). Effects were somewhat larger when removing studies with MHFA developer involvement at post , up to 6-month follow-up , and beyond 6-months .Effects on intentions to provide mental health first aid were moderate-to-large at post-intervention , but small improvements were evident at less than 6-month follow-up . Effects remained similar when removing studies with MHFA founder involvement, post d = -0.14  and follow-up d = 0.27 .There were no improvements in the amount of help provided to a person with a mental health problem at post , but no effects were evident at less than 6-month follow-up . One study found small, non-significant improvements at 12-month follow-up (d = 0.25).The quality of mental health first aid provided to a person with a mental health problem showed a moderate, non-significant improvement at post-intervention (d = -0.04) or by 6-month follow-up (d = 0.16), with no evidence of heterogeneity at either time point. One study showed no effects at 12-month follow-up (d = 0.02). At follow-up less than 6 months, effects were slightly lower when removing three studies that involved the founders of MHFA, d = 0.11 .Improvement in the mental health of MHFA trainees was not evident at post-intervention , and a non-significant negative effect at 12 month follow-up (d = -0.09). At follow-up less than 6 months, removing the one study that involved the founders of MHFA and was at high risk of bias had little impact on the effect size, d = 0.17 .Three studies evaluated the effect of MHFA training on recipients of MHFA (via MHFA trainees), and found a small non-significant effect by 6-month follow-up , and program type (adult versus youth). Full results are presented in p< .001) and follow-up less than 6 months (p = .005), MHFA confidence at post-intervention (p < .001), and amount of MHFA provided at less than 6-month follow-up (p < .001). Studies that included another mental health education intervention as the comparison condition consistently had smaller mean effect sizes than those using a waitlist, no intervention, health education intervention or other form of comparison condition.Where Ib = -0.06, 95% CI: -0.12, 0.00) and studies with a higher proportion of females were associated with lower amounts of MHFA provided to a person with a mental health problem at follow-ups less than 6 months . Given the lack of consistency in effects, potential for Type II errors from the number of analyses conducted, and the observational nature of meta-regression, these findings should be interpreted with caution.For outcomes with at least 3 studies we investigated whether gender and program length were possible sources of heterogeneity in separate meta-regression analyses. There were no consistent effects across outcomes see . Longer This review identified 18 controlled trials evaluating the effectiveness of MHFA training in a variety of settings. Across primary outcomes, there were generally small to moderate improvements at post-training and up to 6 months later. Effects at up to 12-month follow-up were less clear and require further replication. For knowledge outcomes, there is strong evidence that the training improves knowledge about mental health problems, with effects persisting up to a year after training. It also leads to moderate improvements in beliefs about appropriate treatments. Accurate identification of a person with a mental health problem improved up to 6 months later, but effects were smaller immediately after training and it is unclear why this was the case. MHFA training led to approximately small reductions in stigmatising attitudes, with post-hoc analyses suggesting greater reductions in social distance attitudes than other measures of stigma. Perceived confidence in helping a person with a mental health problem was largest post-training, but moderate effects persisted up to 6 months later. At post-training there were moderate-to-large improvements in intentions to provide first aid to a person with a mental health problem, which reduced at follow-up. Perhaps unsurprisingly, there were no changes in the amount of MHFA provided to others at post-training, given the short duration of the course and lack of opportunity to assist others. However, small effects were evident by 6-month follow-up, with heterogeneity explained by the lack of effect in studies comparing MHFA with another mental health education intervention. The impact of MHFA training on the quality of behaviours offered to a person with a mental health problem is less clear, as the pooled effect size confidence intervals failed to rule out no improvement or less improvement compared with controls. Effects on secondary outcomes–the mental health of trainees and MHFA recipients–were less convincing. There were potentially very small effects at up to 6-month follow-up, but estimates were imprecise and may change with further research.Overall, the effects of MHFA training were robust to researcher allegiance, as effects were similar when excluding studies conducted by the developers of MHFA. This suggests that outcomes will be similar if evaluated by different research groups in different countries, and supports the program’s spread worldwide. Several outcomes showed significant variation in effect sizes across studies, which was often explained by the choice of comparison condition. Studies that compared MHFA with a different mental health education intervention generally showed no significant difference in effects. Other subgroup analyses investigating the effect of delivery format and program type were inconclusive due to a lack of power. With the exception of MHFA knowledge, the quality of the evidence was generally moderate or low, typically due to lack of blinding or potential bias from missing outcome data. It is difficult to achieve blinding for this type of intervention, as it requires a plausible control condition. The combination of self-reported outcomes with participants aware of whether they were in the intervention or control meant that most studies suffered potential bias in measurement of outcomes. This limitation could be overcome with an objective measure of MHFA behaviours, such as a simulated encounter with a person with a mental health problem, as has been used to evaluate therapist competence . NeverthResults were generally consistent with those reported in an earlier review of MHFA that included fewer controlled trials  and 6 ovThis review’s strengths were its rigorous methodology and comprehensive inclusion of relevant studies, including 3 unpublished or ongoing studies. We extracted the full range of outcomes from MHFA training and conducted a detailed evaluation of risk of bias of each study outcome. Limitations included the small number of studies in some analyses, which affected the precision of pooled estimates and limited the power to investigate heterogeneity in subgroup and meta-regression analyses.This review has identified some evidence gaps that could be further investigated. Only two studies have thus far examined MHFA training effects beyond 6 months , 47, henAlthough effects were generally small to moderate, MHFA training could potentially have a large public health impact. Evidence suggests that many people are not well informed about how to recognise mental health problems in others, how to respond to them, and what services and effective treatments are available . StigmatIn conclusion, this review supports the effectiveness of MHFA training in improving mental health literacy and appropriate support for those with mental health problems. This public health intervention is a noteworthy contribution to improving the lives of people with mental health problems.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.